Abstract Microtubule-based intracellular transport is critical for the function of all eukaryotic cells. Cytoplasmic dynein is a minus-end-directed microtubule motor that transports a variety of cargoes, and dynein-cargo interactions require the dynactin complex and specific cargo adapters. How dynein-cargo interactions are regulated in vivo and how dynein activity is coordinated with cargo binding are major open questions in the field. Recently, structural studies and in vitro motility assays led to the hypothesis that the binding of cargo to the pointed end of dynactin's Arp1 filament may unlock dynactin's p150 subunit via a change in its conformation, leading to dynein activation. However, in vivo evidence supporting this hypothesis is missing and the identity of proteins involved in p150's conformational change remain to be defined. We previously found that dynactin's pointed-end protein p25 in Aspergillus nidulans is important for dynein- early endosome interaction. Recently, we discovered that p25 is required for the interaction between dynein-dynactin and HookA, the early endosome dynein adapter. Based on our recent data, we hypothesize that p25 is a key protein regulating dynactin conformation. Specific Aim 1 is to reveal the roles of dynactin p25 and the cargo adapter HookA in regulating dynactin. Sensitized emission-based FRET imaging, microtubule pelleting and in vitro motility assays will be used to achieve this aim. In addition, our recent genetic work has uncovered a vezatin-like protein, VezA, as a novel regulator for dynein-cargo interaction. VezA is not a cargo adapter like HookA but affects the accumulation of dynein at the cargo-loading site, the microtubule plus end. How VezA regulates dynein remains a mystery. Specific Aim 2 is to determine the mechanism of VezA action in dynein-mediated vesicle transport. We will use proteomic and genetic approaches to identify new players in the VezA-dynein pathway.